Quantitative analysis of trace amounts of material in a sample (&lt;10.sup.-8 -10.sup.-9 M) requires sensitive and specific detection methods. Otherwise, identification of trace materials in a sample can be masked by substances found at higher concentrations. Direct measurement of trace materials is often difficult in a small sample due to the low concentration of analyte. This problem is compounded if the substance of interest does not have a physical or chemical property (e.g., UV absorbance) that is easily measurable. Introduction of radioisotopic labels into an analyte can provide a sensitive method of detection; however, disposing of large quantities of radioactive waste can be expensive.
Immunochemical detection using antibodies to a target trace material provides one of the most specific methods to identify trace compounds in a sample. Immunoblotting and ELISA procedures utilize an antibody attached to an enzyme which catalyzes the conversion of an added substrate to a colored or fluorescent product which can be detected. However, crossreactivity and non-specific binding can decrease accuracy, and these procedures can require many steps, expensive chemicals, and long incubation times.
U.S. Pat. No. 5,137,609 to Manian et al. discloses an electrophoresis-based detection system for abundant volumes of target analyte using fluorescently labelled binding agents and measurement of differential migration times. Manian et al. calibrate the apparatus by recording the time when free binding agent arrives at a measuring place, then use this time to establish a window in which to expect the complex. Data from recorded signals from the fluorescent labels are then searched to establish the relationship between free binding agent and bound complex.
Nielsen et al. (J. Chrom. 539:177-185 (1991)) discloses separation of formed antibody/antigen complexes by free solution capillary zone electrophoresis. Direct measurement of UV absorbance of the antigen, antibody or complex is used to detect and identify the complexes.